CN101153281A - DNA on-line separating microcurrent control chip and analytical method thereof - Google Patents
DNA on-line separating microcurrent control chip and analytical method thereof Download PDFInfo
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Abstract
The present invention relates to a DNA separation technique, in particular to a micro-fluidic chip for online DNA pre-concentration and separation. The chip consists of a leading buffer pool, a sample pool, a sample waste liquid pool, a buffer waste liquid pool, a terminating buffer pool and channels between the pools, wherein, the terminating buffer pool is arranged at the outer side of the sample pool, and the leading buffer pool is arranged between the sample pool and the buffer waste liquid pool. A separation detection point is arranged near the inside of the buffer waste liquid pool, and a pre-concentration detection point is arranged at the crossing point of an extending channel and a separation channel of the leading buffer pool. The present invention combines online electrophoresis pre-concentration and electrophoresis separation after concentration of DNA samples into the micro-fluidic chip which is of flexible design, simple processing, convenient operation, high sensitivity and extensive application.
Description
Technical field
The present invention relates to the DNA isolation technique, specifically a kind of on the micro-fluidic chip platform, adopt online electrophoresis pre-concentration and concentrated rear electrophoresis separation method to realize the micro-fluidic chip and the analytical procedure thereof of DNA ON-LINE SEPARATION.
Background technology
The separation of DNA and detection are the related important technical of gene studies.The DNA that detects can be amplification or nonamplifie sample.For non-amplification sample,, often need a large amount of tissues or cell because copy is limited; For the amplification sample, because the concentration of the DNA in the actual biological sample is generally lower, even after polymerase chain reaction (PCR) amplification, also can not guarantee the detection sensitivity that reaches enough, the method of addressing this problem is to increase the polymerase chain reaction number of times, and its cost is more reagent and time loss.For traditional microchip electrophoresis and capillary electrophoretic analysis method, can improve detection sensitivity though increase sample size, also cause the reduction of separation efficiency simultaneously.The isotachophoresis pre-concentration is a kind of by increasing that sample size improves detection sensitivity and the effective ways that do not influence separation efficiency.Because the isotachophoresis pre-concentration-separation and combination method required equipment interface based on traditional capillary electrophoresis platform is very complicated, cause bigger dead volume simultaneously and influenced its actual application value, and use the micro-fluidic chip platform can realize this isotachophoresis pre-concentration-separation and combination method easily.Previous separation method not on the microfluidic control chip with pre-concentration and concentrated rear electrophoresis separation and combination, this realization online DNA pre-concentration-isolating micro-fluidic chip and analytical procedure thereof do not have relevant report.
Summary of the invention:
The object of the present invention is to provide a kind of flexible design, processing simple, analyze sensitive more, micro flow controlling chip DNA separates and analytical procedure with separating efficiently fast.
For achieving the above object, the technical solution adopted in the present invention is:
Chip by leading buffer pool, sample pool, sample waste liquid pool, damping fluid waste liquid pool, trail buffer pool and between passage constitute.Wherein, trail buffer pool in the sample pool outside, leading buffer pool is arranged between sample pool and the damping fluid waste liquid pool.Be adjacent to damping fluid waste liquid pool inboard and be provided with the separation detection point, be provided with the pre-concentration check point in the extension passage of leading buffer pool and the place, point of crossing of split tunnel.
Be provided with the sample waste liquid pool trailing between buffer pool and the leading damping fluid; Effective sample area between described sample waste liquid pool and the sample pool is 0.5-20cm.
When the sample waste liquid pool was between sample pool and the leading damping fluid, the effective area was 0.5-15cm between leading damping fluid and the sample waste liquid pool;
Or when sample pool was between sample waste liquid pool and the leading damping fluid, the effective area was 0.5-15cm between leading damping fluid and the sample pool.
The microflow analysis method of DNA ON-LINE SEPARATION micro-fluidic chip:
1) when the sample waste liquid pool is between sample pool and the leading damping fluid, inject leading damping fluid between sample waste liquid pool and the damping fluid waste liquid pool, between sample waste liquid pool and sample pool, inject the sample that to analyze, at sample pool with trail between the buffer pool injection and trail damping fluid;
Or when sample pool is between sample waste liquid pool and the leading damping fluid, inject leading damping fluid between sample pool and the damping fluid waste liquid pool, between sample waste liquid pool and sample pool, inject the sample that to analyze, at the sample waste liquid pool with trail between the buffer pool injection and trail damping fluid;
2) trail buffer pool ground connection this moment, leading buffer pool connects high pressure (voltage is the 100-5000 volt), and preceding drain, sample, following fluid are moved along passage, and promptly can detect sample by the pre-concentration check point this moment, finishes sample concentration; Separate at last, leading buffer pool ground connection, the damping fluid waste liquid pool connects high pressure, and promptly can detect sample by separation detection point this moment.
Described each liquid-filling pool links to each other with electrode, and all online electrophoresis pre-concentration compartment analysis steps are switched by the automatic electrode of routine and controlled.
Anionic travelling speed in the described leading damping fluid is faster than the negatively charged ion in the sample, and leading damping fluid can be: chlorion, phosphate anion or borate ion etc.
Described anionic travelling speed of trailing in the damping fluid is slower than the negatively charged ion in the sample, trails damping fluid and can be: negatively charged ion such as 3-(N-morphine quinoline) propanesulfonic acid, N-(second-hydroxyethyl) piperazine-N-2 ethane sulfonic acid, N-three (methylol) methyl-3-aminopropanesulfonicacid acid etc.
Sample is with fluorochrome label and add sieving medium, and described sieving medium can be hypromellose, hydroxypropylcellulose, Natvosol, Vltra tears, polyvidone, polyvinyl alcohol, polyoxyethylene glycol, polyacrylamide or the agarose of non gel sieving; Described fluorescence dye can be covalent labeling dyestuff or non-covalent labeling dye, and the covalent labeling dyestuff can be: FITC series, BODIPY series, Cy series, rhodamine series, Alexa Fluor series, DABCYL, HEX, JOX, NAP, Oregon Green 488, Pyrene, ROX, TAMARA, TET or Texas red; Non-covalent labeling dye can be: SYBR series, Cyanine series, SYTOX series, Thiazole series or ethidium bromide.
The present invention has following advantage:
1. at same micro-fluidic chip, this micro-fluidic chip flexible design, processing are simple with online electrophoresis pre-concentration and concentrated rear electrophoresis separation and combination in the present invention.
2. easy and simple to handle, highly sensitive.Adopt the analytical procedure of electrophoresis sharp separation of the present invention, the The whole analytical process time, the cross sample injection method of remolding sensitivity routine improved less than 500 seconds.
3. widely applicable.The present invention is applicable to amplification and non-amplified production, comprises the DNA in genomic dna, the various body fluid, synthetic DNA and pcr amplification product.Because DNA research is widely used in clinical and biomedical research, so the present invention has wide usage range, comprises the detection, various based on the detection of specific DNA fragments and the analysis of genomic dna in the biological sample of hybridization of various PCR-based at least.
4. chip of the present invention comprises that two basic functional units constitute: the one, and the online electrophoresis pre-concentration of DNA, the 2nd, the electrophoretic separation analysis after DNA concentrates.All online electrophoresis pre-concentration and compartment analysis step are switched by automatic electrode and are controlled, this integrated form micro-fluidic chip has increased the sample feeding amount, use the discontinuous electro-phoresis damping fluid to finish isotachophoresis pre-concentration process in the pre-concentration process, when significantly having improved detection sensitivity, do not lose resolution.
Description of drawings:
Fig. 1 is an integrated microfluidic chip structure iron of the present invention;
Fig. 2 is the analytical procedure figure of the online pre-concentration of isotachophoresis of the present invention-non gel sieving DNA isolation;
(the micro-fluidic chip analytic sample is: PCR product institute reference numbers is Fig. 3: the base pair number of DNA sample) for the electrophoretogram of non gel sieving DNA isolation of the present invention;
Fig. 4 is that the online pre-concentration of isotachophoresis of the present invention-non gel sieving separates and the electrophoresis of simple non gel sieving DNA isolation compares collection of illustrative plates (institute's reference numbers is the base pair number of DNA standard model among the figure);
Fig. 5 is continuous 5 the operation spectrograms of the present invention;
Number in the figure is: 1. leading buffer pool, and 2. sample waste liquid pool, 3. sample pool is 4. trailed buffer pool, 5. damping fluid waste liquid pool, 6. separation detection point, 7. pre-concentration check point.
Embodiment
The micro-fluidic chip of DNA ON-LINE SEPARATION of the present invention can obtain long effective sample area band, and specific constructive form as shown in Figure 1; Whole online electrophoresis pre-concentration compartment analysis process is controlled automatically with electrode, does not wherein have artificial interference.Can be quartz, glass, PMMA, PDMS polymkeric substance for DNA analysis with the micro-fluidic chip material; Internal surface can modify or unmodified is crossed:
(1) select for use static modifying, dynamic embellishment that chip is carried out pre-treatment (ordinary method that adopts in this area) for quartzy, glass-chip:
The static modifying method is: the surface that organic polymer, DNA is bonded in the micro-fluidic chip passage by coupling agent, organic polymer is polyvinyl alcohol, polyvinylacetate, hydroxyalkyl vinyl Mierocrystalline cellulose, polyoxyethylene glycol, polyacrylamide, glycan, and DNA is a bovine serum albumin.
The dynamic embellishment method is: adding with the tensio-active agent in damping fluid is the additive of main component, and described tensio-active agent is anion surfactant, cats product and amphoterics.
(2) can modify (ordinary method that adopts in this area) for plastic chip or do not modify.
Chemistry, physics two big class methods are selected in the plastic chip finishing for use: physical method is Cement Composite Treated by Plasma, photo-irradiation treatment; Chemical process is hydrophilic, hydrophobicity processing.
For quartzy, glass-chip, its internal surface adopts static modifying, organic polymer is bonded in the surface of micro-fluidic chip passage by coupling agent; For plastic chip, its internal surface adopts injection moulding process chemical substance addition means to carry out finishing.
The online electrophoresis pre-concentration method of the microflow analysis method of DNA ON-LINE SEPARATION micro-fluidic chip: DNA comprises: electrical field magnified injection, sample are piled up, isotachophoresis;
Electrophoresis separating method behind the DNA pre-concentration comprises: zone electrophoresis method, micellar electrokinetic chromatography method, kapillary screening electrophoretic method, capillary isoelectric focusing.
The online electrophoresis pre-concentration method of DNA of the present invention is: isotachophoresis; Electrophoresis separating method behind the DNA pre-concentration is: kapillary screening electrophoresis.
The isotachophoresis process is used the discontinuous electro-phoresis damping fluid.Leading ion can be negatively charged ion such as chlorion, phosphate anion, borate ion, and trailing ion can be negatively charged ion such as 3-(N-morphine quinoline) propanesulfonic acid, N-(second-hydroxyethyl) piperazine-N-2 ethane sulfonic acid, N-three (methylol) methyl-3-aminopropanesulfonicacid acid; The sieving medium of non gel sieving is various forms of macromolecular compounds, as hypromellose, hydroxypropylcellulose, Natvosol, Vltra tears, polyvidone, polyvinyl alcohol, polyoxyethylene glycol, polyacrylamide, agarose etc.
Leading ion of the present invention is a chlorion, trailing ion is N-(second-hydroxyethyl) piperazine-N-2 ethane sulfonic acid (HEPES). sieving medium carries out mark for Vltra tears .DNA with fluorescence dye FITC or SYBRGreen, uses laser-Induced Fluorescence Detection after the concentrating and separating.
With fluorescence dye DNA is carried out mark, used fluorescence dye is covalent labeling dyestuff, non-covalent labeling dye:
The fluorescence dye of DNA covalent labeling is selected from FITC series, BODIPY series, Cy series, rhodamine series, Alexa Fluor series, DABCYL, HEX, JOX, NAP, Oregon Green488, Pyrene, ROX, TAMARA, TET and Texas red;
The fluorescence dye of the non-covalent mark of DNA is selected from SYBR series, Cyanine series, SYTOX series, Thiazole series and ethidium bromide.
The online electrophoresis pre-concentration of DNA-electrophoretic separation is analyzed integrated microfluidic chip by shown in Figure 1, and material is quartz, glass, different organic materials such as PMMA, PDMS etc.1 to No. 5 hole is respectively in order to load leading damping fluid, sample waste liquid, sample, to trail damping fluid and damping fluid waste liquid among Fig. 1.
Be full of the non gel sieving dissociating buffer in the passage of integrated microfluidic chip, trail ionogen and in 1 passage, be full of, between 1 and 4 passages, form the electrolytical interface of different electrophoretic mobilities, carry out isotachophoresis behind the DNA sample feeding and concentrate, concentrate the back and realize that non gel sieving separates.In split tunnel, carry out the compartment analysis of DNA, need carry out various forms of finishinges, to prevent the absorption of DNA in vias inner walls.Modification can be any type of physics or chemically modified.Mainly contain static and dynamic two kinds of modifying method for quartzy and glass-chip modifying method, static modifying typically refers to some organic polymers or some DNA is bonded in the surface of passage by coupling agent, and organic polymer mainly contains polyvinyl alcohol, polyvinylacetate, hydroxyalkyl vinyl Mierocrystalline cellulose, polyoxyethylene glycol, polyacrylamide, glycan etc.Important being meant of dynamic embellishment adds some additives in damping fluid, mainly be some tensio-active agents, comprises anion surfactant, cats product and amphoterics etc.For the finishing of plastic chip, mainly be physics or chemical process, physical method has Cement Composite Treated by Plasma, photo-irradiation treatment etc., and chemical process mainly is to carry out hydrophilic to channel surface or the hydrophobicity processing according to the difference of material.
Separating step is as shown in Figure 2:
I sample introduction: No. 3 electrode groundings, No. 2 electrode output;
Ii trails the dielectric medium perfusion: No. 4 electrode groundings, No. 2 electrode output;
Iii sample concentration: No. 4 electrode groundings, No. 1 electrode output;
Iv separates: No. 1 electrode grounding, No. 5 electrode output;
Integrated form micro-fluidic chip (referring to Fig. 1) is by leading buffer pool 1, sample pool 3, damping fluid waste liquid pool 5 and be close to the inboard separation detection points 6 that are provided with of damping fluid waste liquid pool 5 and form, and trails buffer pool 4 in sample pool 3 arranged outside; Leading damping fluid 1 is arranged at sample pool 3 and damping fluid waste liquid pool 5; Be provided with pre-concentration check point 7 in the extension passage of leading buffer pool 1 and the place, point of crossing of split tunnel.
Be provided with sample waste liquid pool 2 trailing between buffer pool 4 and the leading damping fluid 1.
Effective sample area between described sample waste liquid pool 2 and the sample pool 3 is 20cm.
When sample waste liquid pool 2 was between sample pool 3 and the leading damping fluid 1, the effective area was 15cm between leading damping fluid 1 and the sample waste liquid pool 2;
Described each liquid-filling pool links to each other with automatic motor, and all online electrophoresis pre-concentration compartment analysis steps are switched by the automatic electrode of routine and controlled.Used integrated microfluidic chip adopts standard photoetching, wet etching and bonding to make with glass substrate, and line size is 80 μ m * 30 μ m, and pipeline is modified with linear polyacrylamide.
The microflow analysis method of DNA ON-LINE SEPARATION:
1) leading damping fluid 1 adds leading damping fluid, applying malleation makes leading damping fluid be full of the passage All Ranges, in sample pool 3, add sample afterwards, in trailing buffer pool 4, add and trail damping fluid, add leading damping fluid at leading damping fluid 1 and damping fluid waste liquid pool 5;
2) when voltage is added between sample waste liquid pool 2 and the sample pool 3, between sample waste liquid pool 2 and sample pool 3, inject the sample that to analyze;
3) when voltage is added on sample pool 3 and trail between the buffer pool 4, at sample pool 3 with trail between the buffer pool 4 to inject and trail damping fluid;
4) when voltage is added on leading damping fluid 1 and trails between the buffer pool 4, trail buffer pool 4 ground connection this moment, leading buffer pool 1 connects high pressure (voltage is 500 volts), leading ion, sample, trailing ion are moved along passage, since negatively charged ion in the preceding drain and the anionic travelling speed in the following fluid respectively faster than with the negatively charged ion that is slower than in the sample, make sample at leading buffer pool 1 with trail in the passage between the buffer pool 4 and be concentrated, promptly can detect sample by pre-concentration check point 7 this moment, finishes sample concentration.
5) be to separate at last, leading buffer pool 1 ground connection, damping fluid waste liquid pool 5 connects high pressure, is leading damping fluid in the passage between at this moment leading buffer pool 1 and the damping fluid waste liquid pool 5, and the DNA print obtains separating according to clip size in dissociating buffer.Promptly can detect sample (referring to Fig. 3 DNA isolation electrophorogram) by separation detection point 6 this moment; Described sepn process uses laser induced fluorescence(LIF) that sample is detected.
Described leading damping fluid composition is: 15mM HCL/36mM imidazoles, pH value 7.0, and contain 1 μ mol/L SYBR Green as insert labeling dye and 2%HPMC as sieving medium, SYBR Green labeling dye laser excitation wavelength is 473nm, leading ion is a chlorion;
Trailing the damping fluid composition is 20mM HEPES/40mM imidazoles, pH value 7.2, and trailing ion is HEPES;
Sample is: the PCR product of one 120 base pair, with reference to being: the DNA standard model, the DNA standard model comprises 100,250,500,750,1000, the 2000bp fragment.
Integrated form micro-fluidic chip (referring to Fig. 1) is by leading buffer pool 1, sample pool 3, damping fluid waste liquid pool 5 and be close to the inboard separation detection points 6 that are provided with of damping fluid waste liquid pool 5 and form, and trails buffer pool 4 in sample pool 3 arranged outside; Leading damping fluid 1 is arranged between sample pool 3 and the damping fluid waste liquid pool 5; Be provided with pre-concentration check point 7 in the extension passage of leading buffer pool 1 and the place, point of crossing of split tunnel.
Be provided with sample waste liquid pool 2 trailing between buffer pool 4 and the leading damping fluid 1.
Effective sample area between described sample waste liquid pool 2 and the sample pool 3 is 2.2cm.
When sample pool 3 was between sample waste liquid pool 2 and the leading damping fluid 1, the effective area was 3cm between leading damping fluid 1 and the sample pool 3.
When sample pool 3 is between sample waste liquid pool 2 and the leading damping fluid 1, inject leading damping fluid between sample pool 3 and the damping fluid waste liquid pool 5, between sample waste liquid pool 2 and sample pool 3, inject the sample that to analyze, at sample waste liquid pool 2 with trail between the buffer pool 4 injection and trail damping fluid;
Used micro-fluidic chip designs voluntarily for this laboratory, adopts standard photoetching, wet etching and bonding to make with glass substrate, configuration as shown in Figure 1, channel size is 80 μ m * 30 μ m, passage is modified with linear polyacrylamide.The dissociating buffer leading ion is a chlorion, and trailing ion is N-(second-hydroxyethyl) piperazine-N-2 ethane sulfonic acid (HEPES).Sieving medium is a Vltra tears.The DNA standard model is 11 fragments of 72 to 1382 base pairs.In contrast, adopt the cross posture chip of same channels size to carry out conventional cross sample introduction non gel sieving separation.Laser-Induced Fluorescence Detection, the dyestuff of marker DNA are SYBR Green, and laser excitation wavelength is 473nm, the micro-fluidic chip isotachophoresis that obtains concentrates-non gel sieving separate and the electrophoresis comparison collection of illustrative plates of cross sample introduction non gel sieving DNA isolation as shown in Figure 4.Compare with traditional cross posture chip, this integrated form micro-fluidic chip does not lose resolution because of sample size increases, and except that 271/281 base pair fragment, other fragment all reaches baseline separation, and its resolution is similar to cross posture chip.By the calculating of table 1, to compare with traditional cross posture chip, this integrated form micro-fluidic chip detectability has reduced by 300 times.
Table 1 has or not the comparison of isotachophoresis pre-concentration micro-fluidic chip detectability
| The DNA fragment | The total DNA:2.5pg/ μ of integrated isotachophoresis pre-concentration micro-fluidic chip L | The total DNA:500pg/ μ of no pre-concentration function micro-fluidic chip L | ||||
| Concentration pg/ μ L | Signal to noise ratio | Detectability pg/ μ L | Concentration pg/ μ L | Signal to noise ratio | Detectability pg/ μ L | |
| 72 | 0.03 | 32.33 | 0.003101 | 6.7 | 33.5 | 0.5985 |
| 118 | 0.05 | 73.535 | 0.002235 | 10.9 | 64 | 0.5134 |
| 194 | 0.09 | 165.235 | 0.001635 | 18.0 | 83.5 | 0.6470 |
| 234 | 0.11 | 196.975 | 0.001654 | 21.7 | 91.5 | 0.7122 |
| 310 | 0.14 | 142.155 | 0.003037 | 28.8 | 95.5 | 0.9040 |
| 603 | 0.28 | 479.015 | 0.001753 | 55.9 | 252 | 0.6664 |
| 872 | 0.40 | 807.235 | 0.001504 | 80.9 | 433 | 0.5608 |
| 1078 | 0.50 | 901.405 | 0.001665 | 100.1 | 498 | 0.6028 |
| 1353 | 0.63 | 824.835 | 0.002284 | 125.6 | 567.5 | 0.6639 |
| Mean value | 0.0021 | 0.6521 | ||||
Integrated microfluidic chip (referring to Fig. 1) is by leading buffer pool 1, sample pool 3, damping fluid waste liquid pool 5 and be close to the inboard separation detection points 6 that are provided with of damping fluid waste liquid pool 5 and form, and trails buffer pool 4 in sample pool 3 arranged outside; Leading damping fluid 1 is arranged at sample pool 3 and damping fluid waste liquid pool 3; Be provided with pre-concentration check point 3 in the extension passage of leading buffer pool 3 and the place, point of crossing of split tunnel.
Be provided with sample waste liquid pool 2 trailing between buffer pool 4 and the leading damping fluid 1.
Effective sample area between described sample waste liquid pool 2 and the sample pool 3 is 10cm.
When sample waste liquid pool 2 was between sample pool 3 and the leading damping fluid 1, the effective area was 7cm between leading damping fluid 1 and the sample waste liquid pool 2;
Used micro-fluidic chip designs voluntarily for this laboratory, adopt standard photoetching, wet etching and bonding to make with glass substrate, configuration as shown in Figure 1, channel size is 80 μ m * 30 μ m, passage is modified with linear polyacrylamide, the dissociating buffer leading ion is a chlorion, and trailing ion is N-(second-hydroxyethyl) piperazine-N-2 ethane sulfonic acid (HEPES).Laser-Induced Fluorescence Detection, the dyestuff of marker DNA are SYBR Green, and laser excitation wavelength is 473nm.To total dna content is that 2.5pg/ μ L comprises 11 segmental standard models and carries out continuous 5 times and analyze, and the electrophoresis spectrogram as shown in Figure 5.The relative standard deviation of transition time (RSD) is less than 3%, and peak height relative standard deviation (RSD) is less than 10%.
Claims (7)
1. one kind is used for online pre-concentration of DNA and isolating micro-fluidic chip, have leading buffer pool (1), sample pool (3), damping fluid waste liquid pool (5) and be adjacent to the inboard separation detection point (6) that is provided with of damping fluid waste liquid pool (5), it is characterized in that: trail buffer pool (4) in sample pool (3) arranged outside; Leading damping fluid (1) is arranged between sample pool (3) and the damping fluid waste liquid pool (5); Be provided with pre-concentration check point (7) in the extension passage of leading buffer pool (1) and the place, point of crossing of split tunnel.
2. by the micro-fluidic chip of the described DNA ON-LINE SEPARATION of claim 1, it is characterized in that: be provided with sample pool (3) and sample waste liquid pool (2) between buffer pool (4) and the leading damping fluid (1) trailing.
3. by the micro-fluidic chip of the described DNA ON-LINE SEPARATION of claim 2, it is characterized in that: the effective sample area between described sample waste liquid pool (2) and the sample pool (3) is 0.5-20cm.
4. press the micro-fluidic chip of the described DNA ON-LINE SEPARATION of claim 2, it is characterized in that: when sample waste liquid pool (2) was between sample pool (3) and the leading damping fluid (1), the effective area was 0.5-15cm between leading damping fluid (1) and the sample waste liquid pool (2);
Or when sample pool (3) was between sample waste liquid pool (2) and the leading damping fluid (1), the effective area was 0.5-15cm between leading damping fluid (1) and the sample pool (3).
5. an application rights requires the microflow analysis method of 1 described DNA ON-LINE SEPARATION micro-fluidic chip, it is characterized in that: 1) when sample waste liquid pool (2) is between sample pool (3) and the leading damping fluid (1), inject leading damping fluid between sample waste liquid pool (2) and the damping fluid waste liquid pool (5), between sample waste liquid pool (2) and sample pool (3), inject the sample that to analyze, at sample pool (3) with trail between the buffer pool (4) injection and trail damping fluid;
Or when sample pool (3) is between sample waste liquid pool (2) and the leading damping fluid (1), inject leading damping fluid between sample pool (3) and the damping fluid waste liquid pool (5), between sample waste liquid pool (2) and sample pool (3), inject the sample that to analyze, at sample waste liquid pool (2) with trail between the buffer pool (4) injection and trail damping fluid;
2) trail buffer pool (4) ground connection this moment, leading buffer pool (1) connects high pressure, and leading ion, sample, trailing ion are moved along passage, and promptly can detect sample by pre-concentration check point (7) this moment, finishes sample concentration; Separate at last, leading buffer pool (1) ground connection, damping fluid waste liquid pool (5) connects high pressure, and promptly can detect sample by separation detection point (6) this moment.
6. by the described microflow analysis method of claim 5, it is characterized in that: described each liquid-filling pool links to each other with electrode, and all online electrophoresis pre-concentration compartment analysis steps are switched by the automatic electrode of routine and controlled.
7. by the described microflow analysis method of claim 5, it is characterized in that: the discontinuous buffer solution system that comprises leading damping fluid and trail damping fluid is used in sample pre-concentration-sepn process; Leading damping fluid is positioned at before the sample area band, and anion transport speed wherein is higher than all components in the DNA sample; Trail damping fluid and be positioned at after the sample area band, anion transport speed wherein is lower than all components in the DNA sample.
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| CN113198552A (en) * | 2021-03-29 | 2021-08-03 | 苏州深得源健康科技有限公司 | Micro-fluidic chip for extracting nucleic acid and preparation method and application method thereof |
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